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The beaken were cautiously heated over a free &me to drive off any remaining ammonium salts or organic matter. The soluble material was dissolved in distilled water and filtered into weighed 100-ml. tall-form beakers. A few milliliters of concentrated hydrochloric acid were added and the solutions evaporated to dryness. The residues were cautiously heated to dnve off any moisture, cooled, and weighed. The results obtained (total weight of sodium chloride plus potassium chloride) are tabulated in Table 11.
rate decomposition products. No attempt was made to investigate this matter further. Sulfates are incompatible with the principle involved in this method. They would interfere seriously and if present in the original material this method is not applicable in the present form. LITERATURE CITED
Barber and Kolthoff, J . Am. Chem. Soc., 50, 1625 (1928); 51. 3233 (1929).
DISCUSSION
Perhaps the most attractive feature of the J. Lawrence Smith method of silicate decomposition is the simultaneous removal of most of the nonalkali basic constituents. The hydrofluoricperchloric acid decomposition, as used above, accomplishes the same purpose. The perchlorates of magnesium, iron, and aluminum all form oxides upon thermal decomposition, thus rendering them insoluble. The sodium, potassium, and calcium salts, however, form soluble chlorides which can readily be dissolved away from the insoluble oxides. Inssmuch as the special equipment required for the hydrofluoric-perchloric acid decomposition is a platinum dish, and the operation of grinding an already weighed sample may be avoided, this method has certain advantages over the J. Lawrence Smith method. Examination of Tables I and I1 shows that satisfactory resulb are obtained in all cases except where phosphorus is present in the solution, and one value for Bureau of Standards Sample 91. The presence of phosphorus apparently ties up the alkalies in a form which cannot be extracted out of the solid mass of perchlo-
Vol. 17, No. 9
Berzelius, J. J . , Pogg. Ann., 1, 169 (1824). Breeman, L., Jr., and Scholes, S. R., Bull. Am. Ceram. Soc.. 13, 334 (1934).
Caley and Foulk, J . Am. C h .Soc., 51, 1664 (1929). Green,T. C., ChemBt-Analyst, 16,No. 2, 16 (1927). Japhe, D.,Chimie & industrie, 39,915 (1938). Makinen, E., 2. anorg. Chem., 74, 74 (1912). Marvin, G. G.,and Woolaver, L. B., IND.ENQ.CHEM.,ANAL. ED., 17,474 (1945). Pukall, W., Sprehacrl, 66,231 (1933). Read, E. B., J . Am. CeTam. SOC.,18,341 (1935). Scholes and Weasels, Chemist-Anal&, 25,38 (1936). Shead and Smith, J . Am. Chem. SOC.,53,483(1931). Smith, G.F.,Ibid., 47,762 (1925). Smith, J. L., Am. J . Sei., [3]1, 269 (1871); Am. Chemiut. 1. 404 (1871); Chem. Gazette, 2,252 (1853).
Stevens, R.E., IND. ENG.CHEM., ANAL.ED.,12,413(1940). van Tongeren, W., 2. anorg. allgem. Chem., 218,252 (1934). Willard and Diehl. ”Advanced Quantitative Analvsis”.. DD. 252 ff.. New York. D. Van Nostrind Co.. 1943. Willard,’Liggett, and Diehl, IND.ENQ.CHEM.,ANAL.ED., 14.
__
234 (1942). PWBLXCATXON 99, Research Laboratory of Inorganic Chemistry, M.I.T.
Agar-Streak Method for Assaying Antibiotic Substances SELMAN A. WAKSMAN AND
H. CHRISTINE REILLY
N e w Jersey Agricultural Experiment Station, Rutgers University, N e w Brunrwick,
The agar-streak method for asmying antibiotic substances i s rapid, does not require a sterile sample, permits testing unknown substances against several bacteria or fungi at one time, and can b e used to test substances in nonaqueous solutions. Although it is less precise and less rapid than some other methods, it has marked advantages, especially in screening tests with a large number of organisms and in isolation procedure$ of the sntiblotic rubrtmces.
IT
HA6 long been recognized that the results of assaying bacteriostatic and bactericidal substances depend largely upon the methods employed. Such results may vary considerably, and are iduenced by the species of test organism used, the composition and reaction of the medium, and the time and temperature of incubation. Although standard conditions can be established for accurate evaluation of a single substance or of a crude preparation containing a single antibiotic agent, the results may not be very reliable when several antibiotic substances are compared, since substances may vary greatly not only in their selective action upon different bacteria, but also in their mode of attack upon the same organisms; this is especially true of crude products which may contain either a mixture of widely different types of antibiotic substances or a group of closely related modifications of the same general type of substance. I n selecting a method for measuring quantitatively the activity or potency of an antibiotic substance, it is essential to recognize several pertinent facts, which bear upon the nature and antibacterial properties of antibiotic substances in general. Antibiotic substances are primarily bacteriostatic in their action; they are bactericidal to only a limited degree, although mme Substances may possess marked bactericidal properties.
N. J.
Antibiotic substances are selective in their action; they are able in very low concentrations to prevent the growth of some bacteria, whereas much larger amounts are required to prevent the growth of other bacteria; some bacteria may not be inhibited a t all by a articular substance even in very high concentrations. The conitions for the bacteriostatic activity of different antibiotic substances vary greatly; some are not active at all or their activity is greatly reduced in certain media, because of the antagonistic action of some of the constituents of the media upon the antibiotic substance; others require for their activity the presence of certain specific chemical compounds in order to become effective. The mechanism of action of antibiotic agentg varies; some interfere with bacterial cell division, some affect bacterial respiration, still others interfere with the utilization of essential metabolites by the bacteria, either by replacing or combining with s substance necessary for the nutrltion of the orgamsms. Many antagonistic organisms produce more than one antibiotic substance; the culture filtrate of the organism may differ, therefore, in the nature of its activity from that of the active fractions obtained from it. These facts have an important bearing upon the methods used for measuring the activity of or assaying an antibiotic eubstance. Methods already developed include: 1. The serial dilution or titration method (4, 7) 2. The agar diffusion or cup (cylinder plate) method and its modifications (1, 4, 7 , 8) 3. The agar-dtreak method (IO,f4) 4. The turbidimetric method (3) 5. A variety of other methods (9)
Considerable precision may be obtained with the serial dilution and cup methods. For special purposes, aa for measuring the concentration of the antibiotic in body fluids, special methods may have to be devised.
September, 1945
ANALYTICAL EDITION
The agar-streak method described herein has been in use in this and other laboratories since 1940, but hss been described only briefly (14, 16) in the literature. It is rapid, it does not require a sterile sample, it permite the testing of unknown substances against several bacteria or fungi a t one time, and it can be utilized for testing substances in nohaqueous solutions. It is less precise than the agar diffusion or cup method, the serial dilution method in liquid media, and the turbidimetric method, all of which require separate asaays with each bacterium; it is also leas rapid than some of these methods. The cup method, moreover, is limited to water-soluble diffusible substances, and a standard preparation of the substance must be maintained for reference. The serial dilution method in liquid medium and turbidimetric methods requires sterile samples. Sterilization by heat destroys many antibiotic substances, and sterilization by filters of the Seitz or Berkefeld types may lead to large losses by absorption. The agar-streak method thus makes possible an immediate insight into the nature of the “bacteriostatic spectrum” (9) of a given antibiotic substance long before it has been isolated and chemically identified. This information is of particular importance in the study of new antibiotic substances, or even of known agents when produced by new organisms or under different conditions of culture. Much of the confusion brought about by the use of new names attached to the same substance when isolated from other organisms might have been avoided had the agar-streak method been employed in testing the new agents, because each agent has its own characteristic bacteriostatic spectrum and different substances can be differentiated on the basis of such spectra ( I d , 14). Thus, if a newly isolated substance is tested simultaneously against several organisms, its possible relationship to a n agent already known may become evident. Table 1.
Bacteriostatic Potency of Three Antibiotic Substances Produced b y A. lumigatus ( 1 1 )
Test Organum E . coli
s. aurcu(I
B. sublilis S. lutea
Fumigstin Fumigacin Gliotoxin Dilution unit. b# agar-aheak method 1,200 1,200 6,000 200,000 2,000,000 1,500,000 40,000 100,000 750,000 100,000 1,000,000 2,000.000
Usually the following four organisms are employed, the American Type Culture Collection numbers being given in parenthesis: (1) Escherichia coli (ATCC 9637), to represent the gramnegative group of bacteria; this organism is now used aa a standard test culture in streptothricin and streptomycin assays. (2) Bacillus subtilis (ATCC 6633), to represent the gram-positive rod-shaped bacteria; this organism is used in penicillin and in streptomycin assays, and is highly sensitive to these substances. (3) Staphylococcus aweus (ATCC 6538), to represent the coccus group of organisms; it is used as the standard in penicillin assays. (4) Ban‘llus mycoides (ATCC 6462) is included because of its resistance to certain antibiotic substances, especially penicillin and streptothricin; it can thus be used for differentiation purposes. Different strains of the same organism may show great variation in sensitivity to the same antibiotic substance (IS); hence, only known strains must be employed in order to have the results comparable. Cert>ainother bacteria may occasionally be added to this listfor example, Sarcina lutea, % saprophytic gram-positive coccus, Serratia marcescens, a gram-negative rod-shaped, pigment-producing culture, certain hemolytic streptococci, and others. PROCEDURE FOR AGAR-STREAK METHOD
LIEDIUM. The medium most commonly used consists of 0.5% Difco peptone, 0.5% sodium chloride, 0.3y0Difco meat extract, and 1.5% agar in tap water. This medium is distributed in 10ml. portions in test tubes and autoclaved a t 7-kg. (151b.) pressure for 20 minutes. The pH of the medium is 7.0.
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TESTORGANISMS. E. coli, B. suhtilis, B. m widee, S. aureus, and 8. lutea, obtainable from the American ‘fype Culture Collection, aa well as certain other organisms, may be used. The bacterial rowth from agar-slant cultures, 20 t o 24 hours old, is suspenled uniformly m 6 to 10 ml. of sterile tap water by scrapin the bacterid growth from the agar with a sterile wire loop. kome bacteria, such aa B . mywides, will not prbduce heavy, uniform suspensions. In such cases, the growth is scraped from the agar and broken up aa much aa possible; the larger particles are dowed to settle out, and the supernatant is used for subsequent streaking.
Table
II.
Yield of Chaetomin in Extracts of Culture of C. coehlioder (5) S. aurcuo B . mycoides B . eublilis S. lutea Dilufion unils per gram of preparation
Acetone extract of
80,000 30,000 80,000 100,000 100,000,000 50,000,000 100,000,000 200,000,000 500,000,000 1oo.ooo,ooo 500,000,000 1,000,000.000 300 200 300 a00 50,000,000
20,000,000 50,000,000
100,000.000
DETAILS OF METHOD. The following procedure is used with typical samples of culture filtrates. Each of a series of six sterile 10-cm. (4-inch) Petri dishes is marked off into four or five sectors, and to each of five dishes is added a portion of the solution to be tested, usually 1.0, 0.3, 0.1, 0.03, and 0.01 ml., respectively. None of the sample is added to the sixth, or control, dish. The agar medium is melted cooled t o 45” C., added in 10-ml. quantities to each dish, and mixed thorou hly with the sample by rocking the dish in the hands. After a%out20 minutes, when the agar has solidified, the test organisms are streaked on the plate, each within a marked sector. Three discrete streaks of each organism are made by means of a bent wire needle that will reduce a streak ap roximately 1 cm. wide. The wire is steri{zed in a flame a n a dipped into a uniform suspension of the test organism, and the three streaks are made on the agar without recharging the inoculating needle. The needle is flamed and recharged between plates. The inoculum streaked on the late cannot be strictly controlled, since the volume of liquif applied by the wire needle varies with the viscosity and surface tension of the bacterial suspension. It has also been found more difficult t o produce a uniform streak with a new, smooth wire than with one which h a been in use for some time and haa become slightly roughelied. The actual number of bacterial cells in the suspension will vary somewhat from day to day with the abundance of growth on the agar slant. Although these factors may limit the precision of the method, they do not appreciably affect its usefulness. The plates are incubated at a temperature of 28” C. for 18 t o 20 hours, and the results recorded. The growth of the bacterial cultures on each plate is compared with their corresponding growth on the control plate. The end point is taken as the highest dilution at which growth is completely or almost completely inhibited. The result is expressed in “dilution units”, that amount of material which, added t o 1 ml. of the test medium, will just inhibit the growth of the test organism. Unitage is calculated by dividing the volume of agar in the plate b the amount of the antibiotic substance added to that plate wbch showed the end point. For example, if out of a series of plates the one containing 0.1 ml. of the antibiotic preparation shows no growth of E.. coli, whereaa the late containing 0.03 ml. of the preparation shows normal growti of E. coli, the end point is said to have been reached in the f i s t plate. The concentration of the active substance is thus 10/0.1, or 100 units per ml. of the preparation. Sometimes the end oint lies between succeasive plates of the series. For example, t l e plate containing 0.3 ml. of the active substance may show complete inhibition; the plate containing 0.1 ml. may show normal growth on two streaks and no growth of the test organism on the third streak; the plate containing 0.03 ml. may show normal growth on all three streaks. In this caae, s n i n k olation is made, and the preparation is said to contain 50 or 6O%lution units per ml. of the active substance. Alcoholic solutions of an antibiotic agent may be assayed in the same way, but the volume of 95% ethyl alcohol added to 10 ml. of agar mu’st not exceed 0.3 ml. ; higher concentrations of alcohol are bacteriostatic. The same is true of methyl alcohol, ethyl acetate, and acetone. The maximum is 0.1 ml. per 10 ml. of agar with dioxane, and even leas with pyridine. Pure antibiotic substances, crude concentrates, and experi-
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ferences can be frequently brought out by the agar-streak method, whereas the ma be missed when other method Fungistatic Activity witi a single test organism are emTrichoployed. CryptoAs er phyton 2. Two close1 related substances, mentagrococcus gilfusAntibiotic Antibacterial activity Fusarium clatatus phutea neostreptothricin a n i stre tomycin, can be substance E . coli B. subtilis 8P. 148 598 forman8 differentiated readily y! the fact that Dilution unita per gram the second substance has an activity against B. mycoides similar to t h i t 1,500,000 Actinomycin 15,000 >20,000,000
